Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin

Abstract

A less than adequate therapeutic plan for the treatment of anthrax in the 2001 bioterrorism attacks has highlighted the importance of developing alternative or complementary therapeutic approaches for biothreat agents. In these regards passive immunization possesses several important advantages over active vaccination and the use of antibiotics, as it can provide immediate protection against Bacillus anthracis. Herein, we report the selection and characterization of several human monoclonal neutralizing antibodies against the toxin of B. anthracis from a phage displayed human scFv library. In total fifteen clones were selected with distinct sequences and high specificity to protective antigen and thus were the subject of a series of both biophysical and cell-based cytotoxicity assays. From this panel of antibodies a set of neutralizing antibodies were identified, of which clone A8 recognizes the lethal (and/or edema) factor binding domain, and clone F1, G11 and G12 recognize the cellular receptor binding domain within protective antigen. It was noted that all clones distinguish a conformational epitope existing on the protective antigen; this steric relationship was uncovered using a sequential epitope mapping approach. For each neutralizing antibody, the kinetic constants were determined by surface plasmon resonance, while the potency of protection was established using a two-tier macrophage cytotoxicity assay. Among the neutralizing antibodies identified, clone F1 possessed the highest affinity to protective antigen, and provided superior protection from lethal toxin in the cell cytotoxicity assay. The data presented provides to the ever-growing arsenal of immunological and functional analysis of monoclonal antibodies to the exotoxins of anthrax. In addition it grants new candidates for the prophylaxis and therapeutic treatment against this toxin.

Neutralizing Monoclonal Antibodies Directed against Defined Linear Epitopes on Domain 4 of Anthrax Protective Antigen

“…It is possible, therefore, that the greater affinity of 2-B12 for its target, compared to the affinities of 14B7 and 1-F1, compensates for the fact that 2-B12 binds to a region of PA outside the receptor recognition domain. Several groups have recently documented the importance of antibody affinity in the neutralization of anthrax toxins (1,46).…”

“…This MAb contacts the face of domain 4 that is involved in receptor recognition, and has been proposed to encompass residues 671 to 721. Certainly, additional neutralizing epitopes exist on domain 4 (1,46). In particular, Abboud and Casadevall (1) suggested a linear epitope, immediately adjacent to or possibly overlapping the 14B7 binding site, as the target of neutralizing antibodies.…”

The Major Neutralizing Antibody Responses to Recombinant Anthrax Lethal and Edema Factors Are Directed to Non-Cross-Reactive Epitopes

“…Moreover, several studies also indicate that antibodies to both EF and LF can protect experimental animals from challenge with anthrax toxins or bacteria (1,7,9,19,32,38,39). Therefore, investigations in our laboratory are focused on defining protective responses to LF and EF components of the tripartite anthrax toxin and using this information to design new vaccination approaches that include well-defined determinants of these proteins.…”

The quorum quenching antibody RS2-1G9 protects macrophages from the cytotoxic effects of the Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dodecanoyl-homoserine lactone

“…Furthermore, another advantage of therapeutic mAbs is the predictability of their pharmacodynamic (PD) and -kinetic (PK) properties, a critical aspect in drug development (Reichert, 2003;Reichert and Valge-Archer, 2007). In light of the potent 3-oxo-C 12 -HSL-mediated cytotoxicity, it might be appropriate to think of it as a bacterial toxin and mAbs have successfully been used as neutralizing agents for a variety of bacterial toxins (Casadevall et al, 2004;Nowakowski et al, 2002;Zhou et al, 2007). Here, we have demonstrated that the quorum quenching antibody RS2-1G9 can efficiently protect murine macrophage from the detrimental effects of the P. aeruginosa quorum sensing molecule 3-oxo-C 12 -HSL.…”